This invention relates to new hybrid cell lines and in particular to hybrid cell lines for production of monoclonal antibodies specific to a human receptor for epidermal growth factor (EGF) which can inhibit the growth of human tumor cells that express human EGF receptors, to the antibodies so produced, to therapeutic methods employing the antibodies, and to therapeutic methods employing the antibodies in combination with anti-neoplastic agents.
Control of cell growth is regulated by the interaction of soluble growth factors and cell membrane receptors.
The first step in the mitogenic stimulation of epidermal cells is the specific binding of epidermal growth factor (EGF) to a membrane glycoprotein known as the epidermal growth factor receptor (EGF receptor). (Carpenter, et al., Epidermal Growth Factor, Annual Review Biochem., Vol. 48, 193-216 (1979)). The EGF receptor is composed of 1,186 amino acids which are divided into an extracellular portion of 621 residues and a cytoplasmic portion of 542 residues connected by a single hydrophobic transmembrane segment of 23 residues. (Ullrich et al., Human Epidermal Growth Factor cDNA Sequence and Aberrant Expression of the Amplified Gene in A-431 Epidermoid Carcinoma Cells, Nature, Vol. 309, 418-25 (1986)). The external portion of the EGF receptor can be subdivided into four domains. Recently, it has been demonstrated that domain III, residues 333 to 460, which is flanked by two cysteine domains is likely to contain the EGF binding site of the receptor. (Lax, et al., Localization of a Major Receptor-Binding Domain for Epidermal Growth Factor by Affinity Labeling, Mol. and Cell Biol., Vol. 8, 1831-1834 (1988)). The binding of EGF to domain III leads to the initiation of pleiotropic responses leading to DNA synthesis and cell proliferation.
It has been found in various types of human tumor cells that those cells overexpress EGF receptors. For example, the cancerous cells of bladder tumors have been shown to have a relatively large population of EGF receptors. (Neal et al., Epidermal Growth Factor Receptor in Human Bladder Cancer: Comparison of Invasive and Superficial Tumors, Lancet, Vol. 1, 366-367 (1985)). Breast cancer cells exhibit a positive correlation between EGF receptor density and tumor size and a negative correlation with the extent of differentiation. (Sainsbury et al., Epidermal Growth Factor Receptors and Oestrogen Receptors in Human Breast Cancer. Lancet, Vol. 1, 364-366 (1985); Presence of Epidermal Growth Factor Receptor as an Indicator of Poor Prognosis In Patients With Breast Cancer. J. Clin. Path., Vol. 38, 1225-1228; Epidermal-Growth-Factor Receptor Status as Predictor of Early Recurrence and Death From Breast Cancer. Lancet, Vol.1, 1398-1400 (1987). The tumorigenicity of a series of human vulval epidermoid carcinoma (A431) clonal variants implanted into athymic mice having different levels of EGF receptors was found to correlate directly with the level of expression of the EGY receptor (Santon et al., Effects of Epidermal Growth Factor Receptor Concentration on Tumorigenicity of A431 cells in nude mice. Cancer Res., Vol. 46, 4701-4700 (1986)). Thus, it has been proposed that overexpression of EGF receptors play a role in the origin or tumorigenesis of cancer cells.
The influence of EGF receptor density on the biological behavior of cancer cells may be mediated by the interaction of the receptor with its ligands--namely, EGF or transforming growth factor (TGF). In the majority of cells, when EGF binds to a specific region of the EGF receptor, the cell is mitogenically stimulated. Other tumor cells, such as A431 cells are not mitogenically stimulated by the binding of EGF to its receptors.
Two groups have reported in vivo growth inhibition of tumor A431 cell xenografts in nude mice by binding monoclonal antibodies to the epidermal growth factor receptor of the tumorous cells. Masui et al. demonstrated that treatment with anti-EGF receptor monoclonal antibodies of the IgG2a and IgGI isotype completely prevented tumor formation in athymic mice by subcutaneously implanted A431 cells when treatment was started on the day of tumor cell inoculation. (Masui et al., Growth Inhibition of Human Tumor Cells in Athymic Mice by Anti Epidermal Growth Factor Receptor Monoclonal Antibodies. Cancer Res., Vol. 44 1002-1007 (1984); Mechanism of Antitumor Activity in Mice for Anti Epidermal Growth Factor Receptor Monoclonal Antibodies With Different Isotypes. Cancer Res. Vol. 46 5592-5598 (1986)). Rodeck et al. used a different monoclonal antibody than Masui of the IgG2a isotype which also binds to the EGF receptor of A431 cells to completely inhibit tumor growth of A431 cells xenotransplanted in mice. (Rodeck et al. Tumor Growth Modulation by a Monoclonal Antibody to the Epidermal Growth Factor Receptor: Immunologically Mediated and Effector Cell--Independent Effects. Cancer Res., Vol. 47, 3692-3696 (1987)).
To date, no one, however, has inhibited the in vitro or in vivo growth of human oral epidermoid carcinoma (KB) or human mammary epithelial (184AIN4 and 184AIN4-T--collectively "184") cells. KB and 184 cells are commonly used in studies relating to the EGF-receptor.
KB and 184 cells are substantially different from A431 cells, especially in terms of their growth response to epidermal growth factor. KB and 184 cells are growth stimulated by high concentrations of epidermal growth factor whereas A431 cells are growth inhibited by high concentrations of epidermal growth factor.
Those differences as well as the lack of complete understanding of the mechanism by which the anti-EGF-receptor antibodies inhibit the growth of tumor cells in vivo, prohibit one from accurately determining whether monoclonal antibodies which bind to EGF receptor of A431 cells and demonstrate anti-tumoral activity on A431 cell xenografts in nude mice will also demonstrate antitumoral activity on KB or 184 cell xenografts in nude mice.
Additionally, because human tumor cells are also growth stimulated by epidermal growth factor, KB and 184 cells provide a more representative pattern of responding to EGF than A431 cells, and, in fact, are used as a model for human tumor cells expressing EGF receptors. (Willington et al. J Cell Biol., Vol. 94, 207-212 (1982).
The primary goal in treating tumors is to kill all the cells of the tumor. A therapeutic agent that kills the cell is defined as cytotoxic. A therapeutic agent that merely prevents the cells from replicating, rather than killing the cells, is defined as cytostatic.
Treatment solely with monoclonal antibodies which bind to the EGF receptor merely prevent the cells from replicating, and thus, the monoclonal antibodies act as a cytostatic agent. In order to overcome the monoclonal antibody's cytostatic limitations, monoclonal antibodies specific to the extracellular domain of human epidermal growth factor receptors have been combined with macrophage or mouse complement to yield a cytotoxic response against A431 cells. (Masui et al., Mechanism of Antitumor Activity in Mice for Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies with Different Isotopes, Cancer Research, Vol. 46, 5592-5598 (1986)).
Anti-neoplastic or chemotheropeutic agents administered by themselves, are effective cytotoxic agents. The use of anti-neoplastic agents such as doxorubicin (adriamycin) and cisplatin, for example, are well known in the art. Use of those reagents by themselves, however, are only effective at levels which are toxic or subtoxic to the patient. Cisplatin is intravenously administered as a 100 mg/m.sup.2 dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/m.sup.2 dose one every 21 days.
Bacterial Expression of Antibodies: The prototypical immunoglobulin structure consists of a 150,000 dalton heterodimer composed of two heavy (50,000 daltons each) and two light (25,000 daltons each) chains. Each heavy and light chain pair are covalently attached by a disulfide bond located between the first and second constant domains that joins the carboxy terminal end of the light chain with the heavy chain. The two heavy and light chain pairs are themselves joined together by one or more disulfide bonds, referred to as the hinge region, located between the two heavy chains [1]. Thus, bacterial expression of an entire active immunoglobulin molecule requires, 1) the complex refolding of both heavy and light chains, 2) the concomitant formation of up to 16 disulfide bonds, and 3) the association of protein dimers to form the final divalent molecule.
Initial attempts to produce antibodies in E. coli focused on the expression of entire heavy and light chains, either separately or together in the same cell line [2, 3]. Low levels of expression for both chains were reported in 1984 by two separate groups. Cabilly et al. [2] working with an anti-carcinoembryonic antigen antibody (CEA) reported expression levels of 3% and 0.5% (percent of total cellular protein) for heavy and light chains, respectively. Boss et al. [3] working with an anti-4-hydroxy-3-nitrophenyl acetyl (NP) antibody was able to express the light chain (13% of total protein) in a protease deficient cell line (K12 strain E103S) but the same system only yielded 1% heavy chain. Despite these difficulties with expression levels, both groups reported the first successful recovery of antibody activity from genes cloned and expressed in E. coli.
Specific antigen binding activity was detected by both groups following reduction, denaturation, and refolding (in the presence of redox reagents) of partially purified chains. No active antibody was detected in a mixture of heavy and light chain whole cell extracts, nor observed in a lysate made from cells coproducing the two chains together [2]. Reported recoveries of activity from the refolding procedures range from 3-5% for the anti-CEA antibody down to as low as 0.007% for the anti-NP antibody. Cabilly found similarly low levels of recovery (0.5%) using native anti-CEA antibody subjected to the same denaturation and renaturation procedures [2]. In addition, Boss observed that the majority of active anti-NP material contained truncated heavy chains, suggesting that the shorter peptides were somehow favored during the refolding process [3]. Finally, the actual formation of complete heterodimeric antibodies remains in doubt since no evidence was obtained for divalency by either group.
Fortunately, it is not necessary to express an entire antibody molecule in order to reproduce its antigen-binding capacity. Native antibody protein can be proteolytically degraded under controlled conditions to yield a number of different fragments, some of which retain the full antibody binding capacity. Digestion with the enzyme papain cleaves the heavy chain peptides at a point between the hinge region and the disulfide bond connecting the heavy and light chains. The resulting fragment, referred to as an Fab, is monovalent with respect to its antigen-binding site. The Fab fragment retains an entire light chain, as well as one-half of a heavy chain, with both chains covalently linked by the carboxy terminal disulfide bond.
Inbar et al., [4] used a mouse IgA-myeloma protein (MOPC315) to demonstrate that an Fab fragment could be further cleaved by pepsin digestion, to yield an even smaller antigen binding fragment. This fragment, referred to as an Fv, has an approximate molecular weight of 25,000 daltons and is composed of the amino terminal variable regions of the heavy and light chains (V.sub.H and V.sub.L, respectively) held together by non-covalent bonds. The Fv fragment was shown to retain the same binding specificity for 2,4-dinitrophenyl (DNP) as well as the same affinity (Kd=4.times.10.sup.-7 M) as the intact antibody.
Efficient production of antibody fragments in bacteria would appear to be less difficult for Fvs than for the larger fragments or for complete antibodies. Protein refolding is simplified since each active V.sub.H or V.sub.L chain is required to form a single globular domain stabilized by one intrachain disulfide bond. The association of the two chains in an active Fv requires noncovalent interactions only and occurs with a Kd greater than 10.sup.-8 M for MOPC315 Fv [5].
The work of Hochman et al. [5] predicts that it should be possible to recombine separately expressed MOPC315 V.sub.H and V.sub.L chains to form active Fv molecules in an efficient manner. They used purified MOPC315 Fv, denatured in 8M urea, to isolate individual V.sub.H and V.sub.L chains by DEAE-cellulose chromatography [6]. The inactive V.sub.H and V.sub.L chain components were recombined to form an active Fv following a simple and efficient (80-90% recovery) refolding procedure. In addition, it was shown that active Fv could be recovered efficiently from reduced as well as denatured material [5]. Since it can be anticipated that reduction as well as denaturation will be required to solubilize and purify overexpressed proteins from E. coli, it is useful to note that neither reduction nor denaturation of native MOPC315 V.sub.H and V.sub.L chains prior to refolding prevented efficient recoveries of the native Fv [5].
The results of these early experiments were encouraging to the extent that they confirmed the possibility of producing recombinant antibody molecules in E. coli. Clearly, however, the low levels of expression in combination with low yields of active material indicated that further efforts would be required for efficient bacterial production of antigen-binding proteins.
Bicistronic Constructs: As a result of the inherent difficulties in recovering active whole antibody chains from E. coli, efforts were directed towards the microbial expression and recovery of active Fv or Fab antibody fragments. Success in these efforts was achieved both in yeast and in bacteria. Recovery of active antibody Fv fragments from E. coli has since been reported using several different strategies.
Initial success was achieved by two separate groups who reported the recovery of secreted active antibody fragments from E. coli by co-expressing the two chains of either an Fv [7] or an Fab [8] on the same plasmid. Both bicistronic constructs were characterized by a joint expression of separate heavy and light chain fragment genes under the direction of a single transcriptional unit. This co-expression allows for the synthesis of approximately stoichiometric amounts of both chains. Translation and refolding of each chain occurs in close proximity to each other within the cell. In addition, each peptide coding region has been engineered for secretion by the addition of an amino terminal bacterial leader sequence, directing the expressed products through the inner membrane to the bacterial periplasm. This membrane translocation mimics the processing of eukaryotic protein into the lumen of the endoplasmic reticuluum (ER), a process which occurs normally during the immunoglobulin assembly process in mammalian B cells [1]. The passage of the recombinant proteins across the E. coli membrane was predicted to be functionally analogous to ER transport, facilitating proper refolding and disulfide formation of antibody fragment molecules [7].
Active antigen-binding fragments were, in fact, isolated by both groups either from the periplasm [7] or directly from the culture medium [8]. Skerra and Pluckthun used a bicistronic construct in which bacterial signal sequences for outer membrane protein A (ompA) and alkaline phosphatase (phoA) were fused to synthetic genes encoding the V.sub.H and V.sub.L domains of McPC603, an anti-phosphorylcholine (PC) mouse IgA antibody [9]. Expression was driven by an isopropyl-.beta.-D-thiogalactoside (IPTG) inducible lac promoteroperator. Active Fv fragments could be rapidly purified to homogeneity by phosphorylcholine affinity chromatography of periplasmic fractions. Typical yields were reported to be approximately 0.2 mg of purified Fv fragment per liter of bacterial culture. Measurements of the affinity of the recombinant Fv gave results identical to the corresponding affinity of native McPC603 isolated from mouse ascites (Kd=6-8.times.10.sup.-6 M).
Better et al. [8] reported higher yields (2 mg/L) of active recombinant L6 Fab (a mouse--human chimeric antibody reactive against the human carcinoma cell line C3347) using a S. typhimurium araB (ParaB) promoter to drive the expression of a bicistronic construct containing the full-length L6 light chain and the N-terminal half of the L6 heavy chain (a truncated heavy chain of this type is referred to as an Fd), both preceded by a pectate lyase (pelB) bacterial leader sequence. This construct directed active L6 Fab to the extracellular culture medium from which it could be directly purified using sequential cation-exchange chromatography. Subsequently, the same group reported the successful recovery of active L6 whole antibody as well as Fab fragment from yeast [10].
The bicistronic construct with bacterial leader sequences has since been successfully employed by others, most notably by those involved in the construction of antibody recombinatorial libraries using polymerase chain reaction (PCR) techniques [11-13]. In brief, these libraries are constructed from a large array of individual heavy and light chain fragments, cloned by PCR amplification from a variety of biological sources such as spleen, peripheral blood lymphocyte, and hybridoma cell RNA using antibody-specific generic primers. The heavy and light chain genes are allowed to randomly assort during a subcloning procedure which finally results in the formation of a repertoire of Fab fragments arranged in bicistronic constructs expressed in bacteriophage lambda vectors. These libraries are screened with labeled antigens to identify and isolate novel antibodies. As interesting as this work has been in terms of its potential to replace hybridoma screening for the production of monoclonal antibodies (a somewhat controversial projection, see Winter and Milstein, 1991 [14]), no data has as yet been presented which demonstrates production of either recombinant active Fv or Fab in E. coli in significantly high yields using a bicistronic system.
Single-chain constructs: Architects of single-chain constructs have taken the bicistronic approach to the bacterial expression of antibody fragments one step further by expressing tandemly linked VH and VL genes together as a single protein. This work was pioneered by two separate groups [15, 16] using a similar system, which employs a 15-20 amino acid, neutral peptide linker to fuse the carboxy terminus of a V.sub.H or a V.sub.L gene to the N-terminus of its corresponding partner (see FIG. 20); the order of the two genes appears to be reversible. Bird et al. [16] used a series of custom designed linker sequences based on protein modeling of their projected single-chain Fvs (sFv) while Huston et al. [15] designed a more generic (Gly4, Ser)3 linker which has since been used extensively by other researchers. Both groups used standard E. coli promoter/operator (P/O) systems such as the hybrid, lambda leftward operator/rightward promoter (OL/PR) [16] or the tryptophan P/O [15] to drive the expression of sFv proteins in bacteria. Reported recoveries of active sFv protein were good, ranging from 5-30% of expressed protein for an anti-bovine growth hormone (BGH) sFv [16] to 13% for an anti-digoxin sFv [15].
The anti-digoxin sFv yields were later optimized to 23% and then the basic construct was modified by the N-terminal addition of the coding region for fragment B of staphylococcal protein A which binds to the Fc region of IgG [17]. The resulting bifunctional molecule (FB-sFv) was recovered at very high efficiencies (46%) and was shown to crosslink IgG to digoxin-bovine serum albumin. The successful addition of an effector domain to the amino terminus of an immunoglobulin binding region was entirely novel and has since been repeated with other Ab fragments [18-20].
Fusion of a toxin gene to the carboxy terminal end of an sFv has been reported by Chaudhary et al. [18]. The initial immunotoxin construct joined a sFv specific for the interleukin-2 receptor (anti-Tac) to a fragment of the Pseudomonas exotoxin (PE40) from which the native exotoxin binding domain was removed. The anti-Tac sFv was constructed using a (Gly4, Ser).sub.3 linker and expression of the immunotoxin was driven by the strong IPTG-inducible polymerase-specific T7 promoter [21, 22]. The resulting purified and refolded fusion protein (recovered at 0.2 mg/L) was shown to be highly cytotoxic to IL-2 receptor-bearing human cell lines but not to receptor-negative cells. This group has also reported the successful construction of several new single-chain immunotoxin proteins including one in which the coding region for a truncated form of diptheria toxin (DT) is linked to the N-terminus of the anti-Tac sFv [18]. The DT-anti-Tac sFv was shown to be as active as its anti-Tac-PE40 sFv counterpart and was recovered at significantly higher levels (3-5 mg/L).
Higher levels of recovery (10-12 mg/L, or 20% recovery) of active single-chain Ab have been reported by other researchers using the T7 promoter and a (Gly4 Ser).sub.3 linker to express a sFv specific to the major cellular receptor for human rhinovirus (ICAM-1) [23]. In general, recoveries of active protein from recombinant single-chain Abs (when reported) remain at or below 10 mg/L levels. It is not clear yet whether the apparent limit on recovery levels of most single-chain proteins is a reflection of the level of gene expression, the result of simple peptide to peptide variability, or the inherent limitations imposed by the complexity of sFv refolding.
Separate chain constructs: The expression of V.sub.L and V.sub.H chains in separate bacterial cell lines followed by recombination of purified peptides to form active Fv, is an alternate approach to either the bicistronic or single-chain strategies. Recombinant V.sub.L and V.sub.H peptides can be independently purified, recombined and refolded in vitro in a potentially efficient manner as predicted by the work on native MOPC315 Fv by Hochman et al. [6]. One major advantage of this method of Fv production includes the prospect of high levels of V.sub.H and V.sub.L peptide expression using T7 promoters. In addition, the refolding problem for each separate chain is relatively simple. It is necessary to form only one disulfide bond in a single globular domain. Bond formation in separate chains can be controlled by adjusting protein concentrations downwards during oxidation in order to form only the correct intrachain disulfide bonds. It may be possible with a combination of high levels of protein expression and enhanced refolding efficiencies to greatly reduce the effect of peptide variability on general recoveries of active Fvs.
The first report of active Fv fragments produced by separate chain expression in E. coli was included in an international patent application filed in 1988 [24]. These workers obtained moderately high levels of expression (20-140 mg/L) of mouse immunoglobulin light and heavy chain variable region peptides using an inducible tryptophan promoter/operator in protease deficient host cell lines [24]. Active Fv fragment specific for a hen egg lysozyme epitope (Gloop2) was recovered at 2% levels following partial purification and subsequent refolding of V.sub.H and V.sub.L peptides.
Baldwin and Schultz [25] have reported recovery of DNP-binding activity from a chimeric MOPC315 Fv using recombinant V.sub.L peptides associated with native V.sub.H protein. Moderate levels of V.sub.L expression (10-30 mg/L) were obtained in the form of a V.sub.L fusion protein. The MOPC315 V.sub.L coding sequence was linked via a factor Xa recognition site to the bacteriophage lambda CII protein with expression being driven by the lambda leftward promoter. The yield of V.sub.L protein following factor Xa cleavage and purification was between 5-20% and this purified V.sub.L was efficiently refolded in the presence of native V.sub.H yielding active Fv at between 20-30% efficiencies. Overall yields of active MOPC315 recombinant Fv from starting material (V.sub.L fusion protein) are therefore calculated to be between 1-6%.
Cheadle et al. [26] reported the cloning and expression of both the V.sub.H and V.sub.L of MOPC315 in E. coli using a bacteriophage T7 promoter sequence. The recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active Fv was affinity purified from the chain mixture by specific binding to DNP-Lysine Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified recombinant Fv displayed a binding affinity identical to the native Fv.
Chimeric Fvs specific for 5-dimethylaminonapthalene-1-sulfonyl (Dns) have been produced using bacterially expressed VH peptides recombined with entire native light (L) chains (44). The V.sub.H chains were produced at surprisingly low levels (10 mg/L) using a T7 promoter in a T7 polymerase transient infection system (lambda phage derivative CE6 [27]). The transient T7 expression system is primarily used when the gene product has been demonstrated to be toxic to host cell growth. Purified V.sub.H was recombined with native homologous light chains and active VHL dimers were recovered with efficiencies between 1-6%.